Abstract:

This invention relates to the production and use of pharmaceutical growth
factor compositions with novel characteristics, e.g. improved solubility
and controlled release characteristics under physiological conditions.
Said compositions of one or more precursor proteins of growth factors of
the GDF family provoke morphogenic effects such as for example growth,
differentiation, protection and regeneration of a variety of tissues and
organs, e.g. bone, cartilage, tendons, ligaments, nerves and skin. The
invention can be advantageously used for the healing of
tissue-destructive injuries and for the prevention or therapy of
degenerative disorders.

Claims:

1-32. (canceled)

33. Recombinant mammalian precursor protein or variant thereof
comprisinga) a protease site necessary for proteolytic cleavage and
liberation of a biologically active mature GDF-5 related protein, andb) a
cystine-knot domain with an amino acid identity of at least 70% to the
102 aa cystine-knot domain of human GDF-5 (amino acids 400-501 of FIG.
1/SEQ ID NO 1); characterized in that said precursor protein is
non-glycosylated and produced in prokaryotes.

35. Precursor protein according to claim 33, said protein having an
alanine residue at position 4435 instead of a cysteine. (Monomeric GDF-5
precursor without signal peptide)

36. Precursor protein according to claim 33, said protein further having
an aminoterminal extension of at least seven amino acids.

37. Precursor protein according to claim 33, said protein comprising a
recombinantly introduced continuous stretch of five or more basic amino
acids (arginine, histidine or lysine).

38. Precursor protein according to claim 36, said protein further
comprisinga) a recombinantly introduced protease site intended for
removal of said aminoterminal extension; orb) a recombinantly introduced
site intended for pH-induced or reducing agent-induced removal of said
aminoterminal extension.

39. Precursor protein according to claim 38 a), said protease site
selected from the group consisting of sites for thrombin, enterokinase,
factor xa or sumo protease.

40. A DNA molecule encoding a precursor protein according to claim 33,
characterized in that the triplets AUA (Ile), AGG, AGA, CGG, CGA (Arg),
CUA (Leu), CCC (Pro) and GGA (Gly) within the first 30 codons have been
replaced by alternative triplets of the genetic code encoding identical
amino acids.

41. A DNA molecule encoding a precursor protein of claim 33, characterized
in that stretches of at least two triplets selected from the group
consisting of AUA (He), AGG, AGA, CGG, CGA (Arg), CUA (Leu), CCC (Pro)
and GGA (Gly) within the first 30 codons have been replaced by
alternative triplets of the genetic code encoding identical amino acids.

43. The process according to claim 42, characterized in that said process
comprises a step of transforming a prokaryotic host cell with a plasmid
containing a DNA molecule encoding a precursor protein, characterized in
that the triplets AUA (Ile), AGG, AGA, CGG, CGA (Arg), CUA (Leu), CCC
(Pro) and GGA (Gly) within the first 30 codons have been replaced by
alternative triplets of the genetic code encoding identical amino acids.

44. The process according to claim 42, further comprising solubilization
of inclusion bodies in a solution comprising 3 mM DTT or less.

45. The process according to claim 44, further comprising a direct
refolding procedure which comprises a step of diluting the inclusion body
solution 10 fold or less.

46. Pharmaceutical composition comprising one or more proteins according
to claim 33.

47. Pharmaceutical composition according to claim 46, characterized in
that said composition is soluble at a pH between 6 and 8.

48. Pharmaceutical composition according to claim 47, characterized in
that said composition is soluble at pH 7.

49. Pharmaceutical composition according to claim 46, said composition
further comprising a protease formulation necessary for proteolytic
release of the mature protein.

50. Pharmaceutical composition according to claim 33, said protease
formulation comprising one or more proteases selected from the group
consisting of matrix proteases and subtilisin like proprotein
convertases.

51. Pharmaceutical composition according to claim 49, said protease
formulation providing retarded release of said proteases.

52. Pharmaceutical composition according to claim 49, characterized in
that said are selected from the group consisting of furin, SPC-4 and
SPC-6.

53. Pharmaceutical composition according to claim 49, characterized in
that said protease formulation comprises a combination of either SPC-1
(furin) and SPC-6, SPC-1 (furin) and SPC-4, or SPC-1 (furin) and SPC-7.

54. Protein according to claim 33 in the form of a depot formulation which
is fully activated inside a mammalian body at the target site by
endogenous or co-administered proteases.

55. Pharmaceutical composition according to claim 46 for parenteral
administration.

56. Pharmaceutical composition according to claim 55, wherein said
parenteral administration is an injection or intracerebral infusion.

58. A method for the diagnosis, prevention and/or therapy of diseases
associated with bone and/or cartilage damage, for promoting cartilage
and/or bone formation and/or spinal fusion, for the diagnosis, prevention
and/or therapy of damaged or diseased tissue associated with connective
tissue including tendon and/or ligament, periodontal or dental tissue
including dental implants, neural tissue including CNS tissue and
neuropathological situations, tissue of the sensory system, liver,
pancreas, cardiac, blood vessel, renal, uterine and thyroid tissue, skin,
mucous membranes, endothelium, epithelium, for the induction of nerve
growth, tissue regeneration, angiogenesis, wound healing including
ulcers, burns, injuries and/or skin grafts, for the induction of
proliferation of progenitor cells and/or bone marrow cells, for
maintenance of a state of proliferation or differentiation for treatment
or preservation of tissue or cells for organ or tissue transplantation,
for the treatment of degenerative disorders concerning the joints to
skeletal elements and/or for meniscus and/or spinal/intervertebral disk
repair, for the manufacture of a therapeutic and/or diagnostic
composition for the prevention and/or treatment of neurodegenerative
disorders in a patient in need of such method, comprising administering
to said patient an effective amount of a protein of claim 33.

59. A method according to claim 58, wherein said neurodegenerative
disorder is selected from the group consisting of Parkinson's disease,
Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Multiple
sclerosis and Huntington's disease.

60. A method according to claim 58, wherein said disease associated with
bone and cartilage damage is osteoporosis.

61. A method according to claim 58, wherein the method is for promoting
hair growth.

62. A method for delivering a GDF-5 related precursor protein to a target
site inside of a mammal, comprisinga) a first step of administration of a
protein according to claim 33, andb) a second independent step of
administration of one or more proteases selected from the group
consisting of Trypsin, matrix proteases and subtilisin like proprotein
convertases to the same target site.

63. A method for delivering a GDF-5 related precursor protein to the
central and/or peripheral nervous system of a mammal, comprising
administering to the mammal a protein according to claim 33.

64. A method for systemic delivery of GDF-5 related precursor protein to a
mammalian body, comprising administration to the mammalian body of a
protein according to claim 33.

65. The process according to claim 42, characterized in that said process
comprises a step of transforming a prokaryotic host cell with a plasmid
containing a DNA molecule encoding said precursor protein, characterized
in that stretches of at least two triplets selected from the group
consisting of AUA (He), AGG, AGA, CGG, CGA (Arg), CUA (Leu), CCC (Pro)
and GGA (Gly) within the first 30 codons have been replaced by
alternative triplets of the genetic code encoding identical amino acids.

Description:

[0001]This invention relates to the production and use of pharmaceutical
growth factor compositions with novel characteristics, e.g. improved
solubility and controlled release characteristics under physiological
conditions. Said compositions of one or more precursor proteins of growth
factors of the GDF family provoke morphogenic effects such as for example
growth, differentiation, protection and regeneration of a variety of
tissues and organs, e.g. bone, cartilage, tendons, ligaments, nerves and
skin. The invention can be advantageously used for the healing of
tissue-destructive injuries and for the prevention or therapy of
degenerative disorders.

[0002]A growth factor with a variety of biological attributes is
growth/differentiation factor 5 (GDF-5). The protein is also known as
MP52, very close relatives of GDF-5 with overlapping biological functions
and extremely high amino acid homologies are GDF-6 and GDF-7. The
GDF-5/-6/-7 group is conserved among vertebrate/mammalian species but
does not have known orthologues in invertebrates (Ducy and Karsenty 2000,
Kidney Int. 57, 2207-2214). In general, GDF proteins promote cell
proliferation and differentiation as well as tissue
formation/regeneration and are relevant for a wide range of medical
treatment methods and applications. These dimeric molecules act through
specific receptor complexes that are composed of type I and type II
serine/threonine receptor kinases. The receptor kinases subsequently
activate smad proteins, which then propagate the signals into the nucleus
to regulate target gene expression. Smad independent signalling pathways
are also initiated by these receptors and result in induction of the MAP
Kinase pathway. Smads are a unique family of signal transduction
molecules that can transmit signals directly from the cell surface
receptors to the nucleus, where they regulate transcription by
interacting with DNA binding partners as well as transcriptional
coactivators and corepressors.

[0003]The members of this protein family are initially synthesized as
large precursor proteins which subsequently undergo proteolytic cleavage
at a cluster of basic residues approximately 110-140 amino acids from the
C-terminus, thus releasing the C-terminal mature protein parts from the
N-terminal prodomain. All mature polypeptides are structurally related
and contain a conserved bioactive domain comprising six or seven
canonical cysteine residues which are responsible for the
three-dimensional "cystine-knot" motif of these proteins.

[0004]Within the mammalian body, endoproteolytic cleavage takes primarily
place in the trans-Golgi network. The process finally leads to the
secretion of active mature protein parts, whereas the source material as
well as the prodomain portion of the cleaved precursor protein is
believed to remain in the Golgi compartment.

[0006]Especially the osteogenic properties of GDF-5 have been successfully
used in the past, i.e. to aid the healing of local bone fractures. For
such purposes, combined osteoinductive materials consisting of mature
GDF-5 and solid carrier matrices have been developed (see for example
WO98/21972). However, solid materials are inappropriate for indications
such as i.e. osteoporosis which require a systemic application in order
to guarantee a homogeneous distribution of the protein within the body.
Likewise problematic is a drug delivery to places which are badly
accessible such as the brain or the spinal cord.

[0007]In these cases, administration of GDF-5 in soluble form is generally
preferred. However, the mature protein shows exceptional poor solubility
under physiological conditions. According to this fact, previous attempts
to formulate stable liquid or gel-like GDF-5 compositions have faced
serious problems. A pH-dependent solubility profile of mature GDF-5/MP52
(shown i.e. in EP 1 462 126) reveals that the protein starts
precipitating in aqueous solutions with a pH above 4.25 and becomes
almost insoluble between pH 5 and pH 9. Although EP 1 462 126 succeeded
in improving the protein solubility profile slightly by using solvents
with low ionic strength, high solubility at nearly neutral pH has never
been achieved but is very desirable for parenteral and other
formulations.

[0008]After discovery of their unique tissue inductive activities, growth
factor proteins such as GDF-5 have been successfully applied in
therapeutic research and regenerative surgery, in which they promote and
assist the natural healing process of damaged tissues, either alone or in
combination with specific matrix materials. Nevertheless there is still a
great need to develop novel methods and pharmaceutical compositions for
the efficient administration of such proteins under physiological
conditions, e.g. in cases which do not allow the combination of the
protein with a voluminous solid carrier material. Especially desirable
are formulations from which the active protein is released in a
controlled manner which exactly satisfies the demand of the body.

[0009]It is therefore an object of the invention to improve and facilitate
the medical use of GDF-5 and related proteins by providing liquid growth
factor compositions which are stable, non-toxic and applicable at
physiological pH values. This object comprises the development of
injectable and/or parenteral formulations, controlled release
compositions, and formulations which can be transported across the
blood-brain barrier. A second object of the invention is a method for the
preparation of said formulations and compositions. A third object of the
invention is to provide suitable methods for the local or systemic
administration of said growth factor compositions.

[0010]These objects are solved according to the invention by providing
pharmaceutical compositions containing biologically inactive precursor
proteins related to human Growth/Differentiation Factor 5 (hGDF-5). These
pharmaceutical compositions are intended to serve as controlled release
formulations which are delayed activated inside the mammalian body,
either by endogenous proteases at the target site or by co-administered
proteolytic enzymes.

[0011]Some frequently used terms herein are defined and exemplified as
follows:

[0012]The term "cystine-knot-domain" as used herein means the well known
and conserved cysteine-rich amino acid region which is present in
growth/differentiation factors (GDFs) and which forms a three-dimensional
protein structure known as cystine-knot. In this domain, the respective
location of the cysteine residues to each other is important and is only
allowed to vary slightly in order not to lose the biological activity.
Consensus sequences for cystine-knot domains are known in the state of
the art. According to the definition defined herein the
cystine-knot-domain of a protein starts with the first cysteine residue
participating in the cystine-knot of the respective protein and ends with
the residue which follows the last cysteine participating in the
cystine-knot of the respective protein. For example, the cystine-knot
domain of the human GDF-5 full length (precursor) protein (SEQ ID NO 1)
comprises the amino acids 400-501 (see also FIG. 1).

[0013]The term "precursor protein" as used herein means a biologically
inactive protein comprising a protease site, said site being necessary
for proteolytic cleavage of said precursor protein and subsequently
leading to the release of a biologically active mature protein.

[0014]The term "GDF-5 related precursor protein(s)" as used herein means
any naturally occurring mammalian or artificially created, biologically
inactive precursor protein which comprises a) a protease site which is
necessary for proteolytic cleavage of said precursor protein,
subsequently leading to the release of a biologically active mature
protein, and b) a cystine-knot-domain with an amino acid identity of at
least 70% to the 102 aa cystine-knot domain of human GDF-5 (amino acids
400-501 of FIG. 1/SEQ ID NO 1).

[0016]The term "variant(s)" as used herein means any of the following
polypeptides:

[0017]a) fragments of said protein comprising at least the cystine-knot
domain and the protease site necessary for proteolytic activation.

[0018]b) protein constructs which contain additional sequences in excess
to the original sequence of said protein

[0019]c) any combination of a) and b)

[0020]The term "biological activity" denotes the biological activities of
a GDF-5 related protein or GDF-5 related precursor protein. For example,
this activity can be measured by one or more of the following assays:

[0027]g) measurement of the signal transduction cascade through the
activation of Smads using a reportergene assay based on the
Smad-binding-elements preceding the firefly luciferase gene e.g. are
previously described in Nohe et al., 2002. J Biol Chem. 277, 5330-5338.

[0028]Unlike their mature counterparts, precursor forms of GDF-5 related
proteins are biologically inactive regarding their growth and
differentiation capabilities. In addition, efficient production of these
protein precursors in prokaryotic hosts failed seriously in the past due
to unknown reasons, although in contrast production of the significantly
shorter mature proteins is possible and has been achieved previously (see
e.g. Hotten et al., Biochem Biophys Res Comm 204, 646-652 (1994). Because
of these two facts, addition of GDF-5 related precursors to
pharmaceutical compositions instead of mature proteins never seemed to be
a reasonable option in the past.

[0029]According to the present invention, it has now surprisingly been
found (and it is subsequently demonstrated hereinafter) that specific
sequence modifications in fact allow for the recombinant expression of
GDF-5 related precursor proteins in prokaryotic hosts, a process which is
economically desirable.

[0030]It is furthermore shown that these recombinant proteins, although
expressed in bacteria and therefore lacking essential eukaryotic features
such as e.g. glycosylation, can be proteolytically cleaved and activated
by selected proteases in a manner comparable to glycosylated eukaryotic
precursor proteins. It is also demonstrated that these recombinant
precursor forms, unlike their mature counterparts, are soluble at
physiological pH values and can be used to formulate pharmaceutical
compositions for the therapy of tissue destructive disorders. It is
finally substantiated that the disclosed pharmaceutical compositions
comprising said precursor molecules might be beneficially utilized as
initially inactive controlled release formulations. These formulations
can be parenterally administered and are subsequently activated in situ.

[0031]It is therefore a first object of the present invention to provide
suitable methods for the heterologous recombinant expression of GDF-5
related precursor proteins in prokaryotic host cells such as e.g. E.
coli. Such prokaryotic production is cost effective, efficient and of
considerable commercial interest. Although it is known that mature GDF-5
related proteins are recombinantly producible in bacteria without major
problems, similar attempts to manufacture precursor molecules of these
proteins in sufficient quantities in E. coli failed in the past. It has
been postulated previously that the prodomain of the precursor protein
may comprise partial sequences which are deleterious or even toxic for
bacteria. However, according to the present invention, this explanation
is no longer acceptable because it is shown hereinafter that prokaryotic
expression of the complete precursor sequence is achievable if certain
amino acids are added to the original amino acid sequence. More
precisely, an amino-terminal sequence extension of at least five,
preferably six or seven amino acids, is sufficient to allow bacterial
production of the protein. The following table shows a selection of
tested plasmid/bacterial strain combinations for the expression of GDF-5
related precursor proteins. Note that only constructs comprising a DNA
coding for an aminoterminal basic elongation of the precursor protein led
to the expression of precursor proteins.

[0032]The results disclosed herein demonstrate that said sequence
extension is especially beneficial if it comprises a continuous stretch
of five or more basic amino acids (arginine, histidine or lysine) which
forces the bacterial cell to produce the protein.

[0033]In a preferred embodiment of this part of the invention, the
N-terminus of such fusion protein comprises the sequence HHHHH (5×
histidine). Especially preferred are precursor proteins which comprise a
continuous stretch of 6 histidines. For reasons of precaution it is noted
that said basic stretch is only needed for the protein production within
the bacterial cell and is not required for already established industrial
protein purification techniques.

[0035]If a fusion protein is generally unwanted, it is of course also
possible to modify the original protein sequence without addition of
amino acids, i.e. by simply replacing a part of the original protein
sequence by said continuous stretch of five or more basic amino acids.
However, it is required that such replacement should be done within the
first 10 amino acids of the original protein sequence.

[0036]Non-parenteral administration of a protein comprising an
amino-terminal extension or a modification comprising a basic stretch of
amino acids into mammals might create some immunogenic problems. To avoid
an undesired immune response, it is helpful if the modified or added
amino acid sequence of the bacterially expressed GDF-5 related precursor
protein can be removed prior to the administration to mammalian patients.
Removal can be easily achieved by different techniques, e.g. if an
adequate protease site (which is different from the protease site
required for biological activation) is recombinantly introduced into the
protein sequence of the GDF-5 related precursor protein. In a preferred
embodiment, said protease site is selected from the group consisting of
sites for thrombin, enterokinase, factor Xa or sumo protease.

[0037]As an alternative, the N-terminal extension of the GDF-5 precursor
protein might also be removed by an autocatalytic cleavage process
induced either by pH-shift or reducing agents such as DTT or beta
mercaptoethanol (see example 7). For example, the inducible self-cleavage
activity of protein splicing elements such as e.g. inteins can be used to
separate the GDF-5 precursor protein from the N-terminal affinity tag.

[0038]For this purpose, the GDF-5 precursor protein can e.g. be integrated
in vectors such as the commercially available IMPACT-TWIN (Intein
Mediated Purification with Affinity Chitin-binding Tag-Two Intein, New
England Biolabs) system. IMPACT-TWIN is able to isolate native
recombinant proteins without the use of exogenous proteases. Intein1 is a
mini-intein derived from Synechocystis spdnaB gene and engineered to
undergo pH and temperature dependent cleavage at its C-terminus (Mathys
et al. (1999), Gene 231, 1-13). Intein2 is either a mini-intein from the
Mycobacterium xenopi gyrA gene (pTWIN1) or from the Methanobacterium
thermoautotrophicum rir1 gene (pTWIN2). These inteins have been modified
to undergo thiol-induced cleavage at their N-terminus (Southworth et al.,
(1999) Biotechniques 27, 110-120). The use of thiol reagents such as
2-mercaptoethanesulfonic acid (MESNA) releases a reactive thioester at
the C-terminus of the target protein.

[0039]It has additionally been found that the recombinant expression of
said GDF-5 related precursor protein in prokaryotes can be also enhanced
by other genetic modifications. This enhancement requires that certain
DNA triplets encoding the amino acids isoleucine, arginine, leucine,
proline and glycine, which appear to be detrimental for the expression of
the recombinant precursor proteins in bacteria, have to be removed from a
part of the DNA coding for the GDF-5 related precursor protein.

[0040]More precisely, in another preferred embodiment of this part of the
invention, bacterial expression of a GDF-5 related precursor protein is
clearly facilitated if triplets AUA (Ile), AGG, AGA, CGG, CGA (Arg), CUA
(Leu), CCC (Pro) and GGA (Gly) have been replaced by alternative triplets
of the genetic code encoding identical amino acids. As shown in FIG. 5,
such optimized codon usage has been implemented in a pET15b (T7-Promotor,
Basic-tag) vector system. Expression of the precursor protein in the E.
coli strain Rosetta with optimized codon usage resulted in a high yield
protein production. Use of a similar vector system but without optimized
codon usage in bacterial strain BL21(DE3) yielded lower amounts of the
precursor protein.

[0041]In the most preferred embodiment of this part of the invention, a
continuous stretch of two or more of said detrimental triplets within the
first 30 codons of said DNA molecule has to be avoided.

[0042]The yield and quality of the GDF-5 related precursor protein can be
further dramatically improved if the bacterial production method
comprises optimized purification steps. It has been found out that best
results are achieved if the purification process comprises a direct
refolding step. Direct refolding means that the expressed proteins are
used directly after inclusion body preparation in a refolding procedure
(necessary for renaturation) without prior column purification step.
Usually GDF-5 related proteins are column purified before initiation of
refolding, see e.g. WO96/33215). Especially important for the disclosed
direct refolding procedure is the use of an optimized buffer for
solubilization of inclusion bodies containing not more than 3 mM DTT. It
has been found out that more DTT content interferes with the refolding
procedure, which highly depends on a redox system which is sensitive to
reducing agents like DTT. Due to the low amount of DTT, the refolding
step can be performed in a 1:10 dilution instead of a commonly used 1:100
dilution, thus having positive effects on the protein yield. Specific
parameters of these preferred purification conditions are disclosed in
example 1.

[0043]GDF-5 related precursor proteins which are manufactured in bacteria
by the aforementioned methods have a variety of advantages besides their
cost effective production. It is also essential that the recombinant
precursor proteins of the invention, although expressed in bacteria and
therefore lacking essential eukaryotic features such as e.g.
glycosylation, can be proteolytically cleaved and activated in a manner
comparable to precursor proteins expressed in eukaryotes. Proteolytic
cleavage of proteins belonging to the family of growth and
differentiation factors often occurs at a characteristic RX(K/R)R site
that divides the mature peptide from the amino-terminal prodomain. For
example, the cleavage site of human GDF-5 contains the motif RRKR,
whereas the corresponding sites of GDF-6 and GDF-7 contain the sequence
RRRR. These sites are known to be recognized by subtilisin like
proprotein convertases (SPCs), a family of seven structurally related
serine endoproteases (designated SPC1 to SPC7). Although all
subtilisin-like proprotein convertases can be used for the cleavage of
the precursor proteins of the invention, protease SPC1 (also designated
furin) is especially useful. Also preferred are SPC4, SPC6 and SPC7
because they are coexpressed with growth factor proteins at distinct
sites (see Costam et al. 1996, J. Cell Biol. 134, 181-191). Especially
preferred according to the invention is furthermore the addition of
single SPCs or combinations of different SPCs in pharmaceutical
compositions comprising GDF-5 related precursor proteins. Suitable
combinations are e.g. SPC1 and SPC4, SPC1 and SPC6, and SPC1 and SPC7.
Another preferred option is the cleavage of the precursor proteins with
trypsin (as shown in FIG. 7).

[0044]The extracellullar matrix is believed to serve as storage site for
growth and differentiation factors. Thus, also suitable to release active
mature protein from the precursor proteins of the invention are other
matrix proteases, e.g. matrix metalloproteases, preferably MMP3.

[0045]As a non-limiting example, FIG. 6 and example 3 show the cleavage of
a bacterially produced precursor protein of the invention (human GDF-5
precursor) with proprotein convertase SPC1 (also designated furin). In
mammals, furin is predominantly localized within the trans-Golgi network
(TGN)/endosomal system, but has also been detected on the cell surface
and extracellularly (Molloy et al., 1999).

[0047]The GDF-5-related precursor proteins as defined herein comprise a) a
protease site necessary for proteolytic activation and b) a
cysteine-knot-domain with an amino acid identity of at least 70% to the
102 aa cysteine-knot domain of human GDF-5. Preferred are precursor
proteins comprising a cysteine-knot domain with an amino acid identity of
at least 75%, preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, more preferably at least 95% to the 102 aa
cysteine-knot domain of human GDF-5. However, the limiting value of at
least 70% is well suitable to separate members of the GDF-5/-6/-7 group
of proteins as well as variants thereof from precursors of other proteins
such as other GDFs and other growth factors. A comparison of the 102 aa
cysteine-knot-domains of human GDF-5, human GDF-6 and human GDF-7 (FIG.
2) reveals the high grade of amino acid identity between these proteins.
Human GDF-6 shares 87 (85%) and human GDF-7 83 (81%) identical residues
with the cysteine-knot-domain of human GDF-5. In contrast, GDFs and BMPs
not belonging to the GDF-5/-6/-7 subgroup display much lower identity
values below 60%.

[0048]The determination of corresponding amino acid positions in related
amino acid sequences as well as the calculation of percentages of
identity between can be performed with the help of well known alignment
algorithms and optionally computer programs using these algorithms. The
amino acid identities in this patent application have been calculated by
aligning sequences with the freeware program ClustalX (Version 1.81) with
default parameters and subsequent counting of identical residues by hand.
Default settings for pairwise alignment (slow-accurate) are: gap opening
parameter: 10.00; gap extension parameter 0.10; Protein weight matrix:
Gonnet 250. The ClustalX program is described in detail in:

[0052]ClustalX is a windows interface for the ClustalW multiple sequence
alignment program and is i.e. available from various sources, i.e. by
anonymous ftp from ftp-igbmc.u-strasbg.fr, ftp.embl-heidelberg.de,
ftp.ebi.ac.uk or via download from the following webpage:
http://www-igbmc.u-strasbg.fr/BioInfo/. The ClustalW program and
algorithm is also described in detail in:

[0055]As already explained and shown in example 6 (FIG. 9, respectively),
the recombinant precursor proteins of the invention are biologically
inactive but can be activated in vivo/in situ by proteases. This is
especially advantageous in cases where precursor proteins or
pharmaceutical compositions comprising said precursor proteins are
therapeutically administered to a patient. Since proteases like e.g. SPCs
are detectable in the Golgi network but are also located in other cell
compartments and the extracellular matrix in various amounts in
dependency of the bodies need, they are able to convert administered
protein precursors to active mature proteins by and by in a manner
controlled by the mammalian metabolism. Thus, administration of protein
precursors guarantees a sustained release of the active drug over a
longer period and avoids dosage problems as known from the administration
of mature/active growth factors.

[0056]Although the administration of pure GDF-5 related precursor proteins
should be sufficient for some therapeutic purposes, other indications may
require the administration in combination with protease formulations
comprising e.g. Trypsin, SPCs and matrix metalloproteases, for example
because some tissues are characterized by the lack of endogenous protease
production.

[0057]It should also be mentioned that SPCs and other proteases show
distinct tissue specific expression patterns. It is well known that some
are ubiquitously present whereas others are restricted to a certain
tissue. Although the precursor proteins of the invention can be activated
e.g. by SPC1/furin alone (see example 3), the tissue specific expression
should be taken into account if pharmaceutical compositions comprising
precursor proteins in combination with proteases are administered. In
some cases, precursor protein activation in some tissues may be enhanced
if the pharmaceutical compositions comprise a protease with strong
expression pattern in said tissue. In other cases, two or more proteases
are beneficial for full activation. For example, it is known that SPC2,
SPC3 and SPC4 expression is largely confined to neuronal tissues, whereas
GDF-5 is activated in joints by a combination of two SPCs, namely SPC1
(Furin) and SPC6. SPC4 and SPC6 expression in developing limbs overlaps
with that of Growth and differentiation factors. SPC7 seems to be
ubiquitously expressed.

[0059]In an especially preferred embodiment of this part of the invention,
a pharmaceutical composition comprising a precursor protein and a
protease formulation is designed in a way which provides retarded release
of said proteases. This composition ensures that the precursor proteins
of the invention are activated at a time point when the administered
solution reaches the target site. There are countless methods for the
sustained release of proteins described in the art which can be used. For
a review see e.g. Handbook of Pharmaceutical Controlled Release
Technology (Wise, D., ed.), 2000. For example, a slow-release formulation
may comprise proteins bound to or incorporated into particulate
preparations of polymeric compounds (such as polylactic acid,
polyglycolic acid, etc.) or liposomes.

[0060]In another embodiment of this part of the invention, the protease
formulation is administered separately a certain time period after
administration of the precursor proteins of the invention.

[0061]A prerequisite for pharmaceutical compositions which are intended
for parenteral administration is a nearly physiological pH of said
compositions. Whereas mature GDF-like proteins such as GDF-5 show
exceptional poor solubility under physiological conditions, the
bacterially expressed precursor proteins of this invention demonstrate a
pH-dependant solubility profile which allows for the direct parenteral
administration to mammals. As exemplified in FIG. 8 and Example 5, they
exhibit excellent solubility between pH 6 and pH 8, and especially
important at or around physiological pH 7.

[0062]Whereas mature GDF-5 related proteins are insoluble at physiological
pH and the use of these proteins is therefore restricted to local
delivery, e.g. in combination with solid matrix materials, the excellent
solubility predetermines the precursor proteins and pharmaceutical
compositions of the invention for systemic delivery purposes. This allows
for the efficient treatment of disorders and diseases with systemic
character. The most prominent examples for such a systemic disease are
osteoporosis, rheumatism and osteoarthritis. However, liquid
pharmaceutical compositions such as those disclosed herein can also be
efficiently used for local delivery, e.g. via injection. For example, the
pharmaceutical compositions and precursor proteins of the invention are
useful for the treatment of neurodegenerative disorders such as
Parkinson's disease (e.g. via intracerebral infusion or intranasal
delivery), for the treatment of local osteoarthritis or arthrosis (e.g.
via injection into the affected tissue, organ or joint), for the
regeneration of meniscus and spinal disks (e.g. via injection), for the
treatment of hair loss and skin aging (e.g. via topical creams) etc. . .
.

[0063]According to this novel characteristic, the precursor proteins and
pharmaceutical compositions of the inventions can be administered
parenterally. Such parenterally administered therapeutic compositions are
typically in the form of pyrogen-free, parenterally acceptable aqueous
solutions comprising the pharmaceutically effective component(s) in a
pharmaceutically acceptable carrier and/or diluent. Said parenteral
administration might e.g. be dermal, ocular, pulmonary, topical or
intranasal administration, injection or infusion.

[0064]Of course this invention also comprises pharmaceutical compositions
containing further substances like e.g. pharmacologically acceptable
auxiliary and carrier substances. The formulation may include
antioxidants, preservatives, colouring, flavouring and emulsifying
agents, suspending agents, solvents, fillers, bulking agents, buffers
such as phosphate buffered saline (PBS) or HEPES, delivery vehicles,
excipients and/or pharmaceutical adjuvants. For example, a suitable
carrier or vehicle may be water for injection, physiological saline
solution, or a saline solution mixed with a suitable carrier protein such
as serum albumin. A preferred antioxidant for the preparation of the
composition of the present invention is ascorbic acid.

[0066]The solvent or diluent of the pharmaceutical composition may be
either aqueous or non-aqueous and may contain other pharmaceutically
acceptable excipients which are capable of modifying and/or maintaining a
pH, osmolarity, viscosity, clarity, scale, sterility, stability, rate of
dissolution or odour of the formulation. Similarily other components may
be included in the pharmaceutical composition according to the present
invention in order to modify and/or maintain the rate of release of the
pharmaceutically effective substance. Such modifying components are
substances usually employed in the art in order to formulate dosages for
parenteral administration in either unit or multi-dose form. The finally
formulated pharmaceutical and/or diagnostic composition prepared
according to the present invention may be stored in sterile vials in form
of a solution, suspension, gel, emulsion, solid or dehydrated or
lyophilized powder. These formulations may be stored either in a
ready-to-use form or in a form, e.g. in case of a lyophilized powder,
which requires reconstitution prior to administration. The above and
further suitable pharmaceutical formulations are known in the art and are
described in, for example, Gus Remington's Pharmaceutical Sciences (18th
Ed., Mack Publishing Co., Eastern, Pa., 1990, 1435-1712). Such
formulations may influence the physical state, stability, rate of in vivo
release and rate of in vivo clearance of the pharmaceutically effective
component.

[0067]The pharmaceutical composition may comprise a matrix material, i.e.
in cases where regeneration of bone or cartilage is intended. It is
advantageous to the precursor proteins when they are applied in and/or on
a biocompatible matrix material. Matrix material as used herein means a
carrier or matrix acting as a scaffold for cell recruitment, attachment,
proliferation and differentiation and/or as a potential delivery and
storage device for precursor proteins. In contrast to the solid matrices,
carriers consist of amorphous materials having no defined surfaces and
lacking a specific shape, i.e. alkylcelluloses, pluronics, gelatins,
polyethylene glycols, dextrins, vegetable oils, sugars and other liquid
and viscous substances.

[0068]Uses of growth factors in combination with matrix materials are
extensively published and described, such as for example in WO98/21972.
These matrix materials are equally suitable for precursor proteins of
growth factors according to the present invention. The matrix material
can be transplanted into the patient, e.g. surgically, wherein the
protein or the DNA encoding the protein can be slowly released from the
matrix material and then be effective over a long period of time. All
types of matrix materials are useful in accordance with the present
invention, as long as they are biocompatible and selected for the
intended area or indication of use. The matrix material can be a natural
material, a modified natural material as well as a synthetic material.
All already known matrices for morphogenetic proteins are encompassed.
Examples of natural materials are e.g. autologous, heterologous or
xenologous bone materials, collagen, e.g. collagen type I and III, or
metals like titanium. Also other components of the extracellular matrix
can be used. The extracellular matrix comprises for example the various
collagens, as for example types I, II, V, IX, X, XI and XIII, further
proteoglycanes and glycosaminoglycanes, as for example
chondroitinsulfate, biglycane, decorine and/or hyaluronic acid, or
noncollagenous proteins as for example osteopontin, laminin, fibronectin,
vitronectin, thrombospondin, cartilage matrix protein and dentin
phosphoprotein. All mentioned natural materials may also be used in
artificially modified forms. Examples of modified natural materials are
demineralized bone, thermoashed bone mineral, sintered bone or chemically
crosslinked hyaluronic acid (hydrogel), or metal alloys. Examples of
synthetic materials are polymers like polyglycolic acid, polylactide and
polylactide derivatives such as e.g. polylactic acid,
poly(lactide-co-glycolide), polylactid acid-polyethylene glycol or
glycolide L-lactide copolymers, further polyphosphates, polyethylene
glycol, polyoxyethylene polyoxypropylene copolymers or materials
containing calcium phosphates such as beta-tricalcium phosphate
(Ca3(PO4)2), alpha-tricalcium phosphate and hydroxyl apatite.
Further examples of other useful matrix materials belonging to one of the
above mentioned groups are Ca(OH)2, coral, natural bone mineral, chitin,
non-demineralized bone particles, ceramic bone particles, ceramic dentin,
irradiated cancellous bone chips, plaster of Paris, bioactive glass,
apatite-wollastonite-containing glass ceramic. Also a combination of the
above mentioned carriers and/or matrices can form the matrix material as
for example the combination of hydroxy apatite and collagen (e.g. Healos,
previously available from Orquest, Inc., CA, USA, [now DePuy Acromed, MA,
USA]), a combination of polyglycolic acid and polylactic acid or
polylactid derivatives, or coral-collagen composites. For a non limiting
list of useful carriers and matrices see further i.e. Kirker-Head 2000,
Advanced Drug Delivery 43, 65-92.

[0070]Mature GDF-5 is also useful in wound repair of any kind. It is also
beneficial for promoting tissue growth in the neuronal system and
survival of e.g. dopaminergic neurons. In this context, GDF-5 can be used
for treating neurodegenerative disorders like e.g. Parkinson's disease
and possibly also Alzheimer's disease or Huntington chorea tissues (see
for example WO 97/03188; Krieglstein et al., (1995) J. Neurosci Res. 42,
724-732; Sullivan et al., (1997) Neurosci Lett 233, 73-76; Sullivan et
al. (1998), Eur. J. Neurosci 10, 3681-3688). GDF-5 allows to maintain
nervous function or to retain nervous function in already damaged
tissues. GDF-5 is therefore considered to be a generally applicable
neurotrophic factor. If e. g. nerve guide carriers are coated with GDF-5
or precursor proteins thereof, significant healing of nerve damages of
the peripheral nervous systems can be anticipated.

[0071]The precursor proteins and pharmaceutical compositions of the
invention can also be used for prevention or therapy of damage of
periodontal or dental tissue including dental implants, neural tissue
including CNS tissue and neuropathological situations, tissue of the
sensory system, liver, pancreas, cardiac, blood vessel, renal, uterine
and thyroid tissue, skin, mucous membranes, endothelium, epithelium, for
promotion or induction of nerve growth, tissue regeneration,
angiogenesis, wound healing including ulcers, burns, injuries or skin
grafts, induction of proliferation of progenitor cells or bone marrow
cells, for maintenance of a state of proliferation or differentiation for
treatment or preservation of tissue or cells for organ or tissue
transplantation; for integrity of gastrointestinal lining, for treatment
of disturbances in fertility, contraception or pregnancy.

[0073]Diseases concerning sensory organs like the eye are also to be
included in the preferred indication of the pharmaceutical composition
according to the invention. As preferred neuronal diseases again
Parkinson's and Alzheimer's diseases can be mentioned as examples.

[0074]The following non-limiting examples together with the figures and
sequence protocols are intended to further illustrate the invention.

[0087]FIG. 9 shows the activation of GDF-5 precursor protein in chicken
micromass cultures as described in example 6. The induction of cartilage
production is indicated by the increase in Alcian blue staining. Both
micromass cells incubated with mature rhGDF5 as well as with rhGDF5
precursor protein showed a massive increase of cartilage, indicating the
in vivo cleavage/activation of the recombinant precursor protein outside
of the trans-golgi network.

Analysis of rhGDF-5 Full Length Protein Expression by Western Blotting

[0089]E. coli strains BL21 (DE3) and Rosetta were transformed with the
plasmid pET15b-rhGDF-5 full length. Protein expression was induced with
IPTG. Bacterial pellets were dissolved in SDS sample buffer and separated
under reducing conditions on a 16% acrylamid SDS gel. The proteins were
blotted onto PVDF membrane and detected with a chemiluminescence
detection kit (Applied Biosystems), using the polyclonal antibody Anti
rhGDF-5 (Chicken B Pool).

[0090]Protein expression of rhGDF-5 precursor protein (GDF-5pro) could
only be detected after IPTG induction. In comparison to the BL21(DE3) an
increased protein expression could be achieved in the Rosetta strain. The
improved protein expression in Rosetta might be due to the optimized
codon usage. Protein expression was optimized with different expression
vector system and different E. coli strains. Surprisingly, precursor
protein expression was only possible when rhGDF-5 precursor had an
additional N-terminal protein tag e.g. histidine tag. Protein expression
of rhGDF-5 full length without N-terminal modification was not possible.

EXAMPLE 3

Cleavage of GDF-5 Precursor Protein by Furin

[0091]The in vitro digestion of rhGDF-5 precursor protein was performed
with the specific proprotein convertase furin. Furin cleaves the amino
acid recognition sequence R-K-R-R within the rhGDF-5 full length. A
typical cleavage experiment was carried out with 3 μg rhGDF-5 full
length dissolved in 1× PBS supplemented with 1 mM CaCl2 and
incubated with 3 U Furin (New England Biolabs) at 30° C. over
night. The digestion was controlled by Coomassie stained SDS gels and
Western blot analysis with antibodies directed against the mature
rhGDF-5. RhGDF-5 full length was digested with furin and separated under
non-reducing conditions on a 10% acrylamid SDS gel (see FIG. 6). The
proteins were further blotted onto PVDF membrane and detected via western
blotting with a chemiluminescence detection kit (Applied Biosystems),
using the mouse monoclonal antibody aMP-5.

[0092]The rhGDF-5 full length has a molecular weight of ca. 100 kDa. After
digestion with furin the mature GDF-5 was released. In the western blot,
aMP-5 antibody only detects correctly folded GDF-5, therefore it is
demonstrated that furin generates mature rhGDF-5 from its natural
precursor protein rhGDF-5 full length.

EXAMPLE 4

Measurement of the Biological Activity of rhGDF-5 Precursor Protein In
Vitro by ALP Assay

[0093]5×105 cells of mouse stromal MCHT-1/26 cells were
incubated for 3-4 days in 20 ml cell culture medium (alpha-MEM,
Penicilline/Streptomycine, 2 mM L-glutamine, 10% FCS) at 37° C.,
5% CO2, H2O-saturated. The cells were subsequently washed with
PBS (phosphate buffered saline), trypsinated and resuspended in culture
medium to a density of 3×104 cells/ml. 150 μl were
transferred to each well of a 96 well culture plate and incubated for 24
h at 37° C., 5% CO2, H2O-saturated. After washing with
medium the wells were filled with 120 μl of new culture medium. 40
μl of different dilutions of rhGDF-5 full length and rhGDF-5 for
standard curve (dissolved in 10 mM HCl and diluted at least 250 fold in
medium) were added, followed by another incubation step for 72 h at
37° C., 5% CO2, H2O-saturated. After washing with PBS,
150 μl of lysis solution (0.2% Nonidet P40, 0.2 g
MgCl2×6H2O, adjusted to 1000 ml with water) was added,
followed by 15-18 h incubation at 37° C., 5% CO2,
H2O-saturated. 50 μl of each well were subsequently transferred
to a new 96 well plate. 50 μl of substrate solution (2.5×
concentrated diethanolamine substrate buffer+148 g/I PNPP (sodium
p-nitrophenyl-phosphate) was then added to each well and the plates were
incubated for another 60 min at 37° C., 5% CO2,
H2O-saturated. The ALP-reaction was stopped afterwards with 100
μl of 30 g/I NaOH and finally the optical density was measured with an
automatic microplate reader at 405 nm under consideration of blank value
subtraction. As an example, results (average values of 3 independent
experiments) regarding rhGDF-5 precursor protein either digested with
furin or undigested are shown in FIG. 7. Six different protein
concentrations (14.6 ng/mL, 44.5 ng/mL, 133.2 ng/mL, 400 ng/mL an 1200
ng/mL) have been used in this assay. The undigested rhGDF-5 precursor
protein protein exhibits nearly no biological activity. In contrast
rhGDF-5 full length digested with furin, trypsin or MMP-3 exhibit
biological activity in a dose dependent manner. The protease furin alone
(as a control) has no negative influence on the ALP assay. Therefore we
can conclude that rhGDF-5 precursor protein is a pro-form exhibiting no
detectable biological activity in the alkaline Phosphatase assay. ALP
induction of rhGDF.5 precursor protein depends on a proteolytical
activation.

EXAMPLE 5

Solubility of rhGDF-5 Precursor Protein

[0094]Chromatography purified rhGDF-5 full length was eluted from a size
exclusion column in 100 mM Tris HCl, 5 mM EDTA, pH 8.0 buffer. To
determine the solubility protein solution (1.2 mg/mL) was adjusted with
HCL to pH 2.0 and pH 7.0. Subsequently the protein solutions were
centrifuged 13.000 g for 10 minutes. The supernatant was carefully
removed and the pellet was solved in 10 μl SDS sample buffer. The
supernatant and the pellet were separated on a 10% acrylamid SDS gel and
analyzed by gel densitometry (Aida, Version 3.51)

[0095]The rhGDF-5 full length showed a solubility of 99% in a buffer
comprising of 100 mM Tris HCl, 5 mM EDTA, for the pH values 2, 7, and 8.
As an example for buffer with pH 7.0, 1.03 μg/100 μl protein was
found in the pellet and 98 μg/100 μL was found in the supernatant.
Therefore it can be concluded that rhGDF-5 full length is soluble at a
physiological pH.

EXAMPLE 6

Mimicked In Vivo Activation of Protein Precursors

[0096]The micromass system is intended to mimic the initial conditions
that lead to cartilage deposition in vivo. Primary cultures of
undifferentiated mesenchyme cells of limb buds reproduce cartilage
histogenesis, a fundamental step in the morphogenesis of the skeleton.
Thus, in the micromass system, limb bud cells will form foci of
differentiating chondrocytes. In order to determine a potential cleavage
of the rhGDF-5 precursor protein and formation of biologically active
mature protein outside the trans-Golgi network, we used chicken micromass
cultures and measured cell differentiation and cartilaginous matrix
production.

[0097]Micromass cultures were prepared as described previously (Lehmann et
al., Proc. Natl. Acad. Sci. U.S.A. 100 (2003), 2277-12282) with minor
modifications. Briefly, fertilized chicken eggs were obtained from
Tierzucht Lohmann and incubated at 37.5° C. in a humidified egg
incubator for about 4.5 days. Ectoderm was removed, and cells were
isolated from the limb buds at stage HH23-24 by digestion with 0.1%
collagenase type Ia and 0.1% trypsine. Micromass cultures were plated at
a density of 2×105 cells/10-μl drop. Cells were stimulated
with the proteins rhGDF-5 and rhGDF-5 precursor protein (proGDF5)
respectively; increasing protein concentrations ranging from 0 to 18 nM
were applied. Culture medium (DMEM-F12, 2% chicken serum, 4 mM
I-glutamine, 1000 U/ml penicillin, and 100 μg/ml streptomycin) was
replaced every 2 days. Alcian blue incorporation into the extracellular
matrix of micromass cultures reflecting the production of
proteoglycan-rich cartilaginous matrix measured at day 4 was quantified
after extraction. Alcian blue staining was performed by fixing micromass
cultures at day 4, then incubating with 0.1% Alcian blue, pH 1,
overnight. Quantification of the staining was achieved after extensive
washings with water by extraction with 6 M guanidine-HCl for 8 hours at
room temperature. Dye concentration was determined spectrophotometrically
at A595.

[0098]As a result, both micromass cells incubated with mature rhGDF5 as
well as with rhGDF5 precursor protein showed a massive induction of
cartilage production as indicated by the increase in Alcian blue staining
(FIG. 9), indicating the in vivo cleavage/activation of the recombinant
precursor protein outside of the trans-golgi network.

EXAMPLE 7

Removal of Aminoterminal Protein Extensions

[0099]The N-terminal extension of the GDF-5 precursor protein are
removable by proteolytic processing with proteases such as thrombin,
Factor Xa, enterokinase etc. Alternatively the N-terminal extension can
be eliminated by autocatalytic cleavage processes induced either by
pH-shift or reducing agents such as DTT or beta mercaptoethanol. For this
purpose the GDF-5 precursor protein could be integrated in to the
IMPACT-TWIN (Intein Mediated Purification with Affinity Chitin-binding
Tag-Two Intein) system (New England Biolabs). This system utilizes the
inducible self-cleavage activity of protein splicing elements termed
inteins to separate the GDF-5 precursor protein from the N-terminal
affinity tag. These inteins have been modified to undergo thiol-induced
cleavage at their N-terminus. The use of thiol reagents such as
2-mercaptoethanesulfonic acid (MESNA) releases a reactive thioester at
the C-terminus of the target protein.

[0100]7a) pH Induced Cleavage:

[0101]The rhGDF-5 precursor protein was cloned into a appropriate vector
(e.g. pTWIN, containing the Ssp DnaB self-cleavable intein-tag). The
resulting plasmid was transformed into an applicable E. coli host strain
(i.e. ER2566, BL21). The cells were grown at 37° C. until an OD600
of 0.5-0.7 was reached, protein induction was induced with IPTG. For
column protein purification a chitin column was equilibrated with Buffer
B1 (20 mM Tris-HCl, pH 8.5, 100 mM NaCl, 1 mM EDTA). The cells were lysed
in Buffer B1 and the clarified cell extract was slowly applied to the
chitin column. The column was washed with Buffer B1 to remove the unbound
proteins. The on-column cleavage of the intein-tag was induced by
equilibrating the chitin resin in Buffer B2 (20 mM Tris-HCl, pH 7.0, 100
mM NaCl, 1 mM EDTA). To allow cleavage, the reaction was carried out
overnight at room temperature. Finally the protein was eluted from the
column.

[0102]7b) Thiol-Induced Cleavage:

[0103]The rhGDF-5 precursor protein was cloned into a appropriate vector
(e.g. pTWIN, containing either the Mxe GyrA or Mth RIR1 intein
self-cleavable intein-tag). The resulting plasmid was transformed into an
applicable E. coli host strain (i.e. ER2566, BL21). The cells were grown
at 37° C. until an OD600 of 0.5-0.7 was reached, protein induction
was induced with IPTG. For column protein purification a chitin column
was equilibrated with Buffer B2 (20 mM Tris-HCl, pH 7.0, 100 mM NaCl, 1
mM EDTA). The cells were lysed in Buffer B2 and the clarified cell
extract was slowly applied to the chitin column. The column was washed
with Buffer B2 to remove the unbound proteins. The on-column cleavage of
the intein-tag was induced by equilibrating the chitin resin in Buffer B3
(20 mM Tris-HCl, pH 8.5, 100 mM NaCl, 40 mM DTT, 1 mM EDTA). To allow
cleavage, the reaction was carried out overnight at room temperature.
Finally the protein was eluted from the column with Buffer B3.